0041-1337/02/7407-987/0 TRANSPLANTATION Vol. 74, 987–994, No. 7, October 15, 2002 Copyright © 2002 by Lippincott Williams & Wilkins, Inc. Printed in U.S.A. NONINVASIVE DIAGNOSIS OF BK VIRUS NEPHRITIS BY MEASUREMENT OF MESSENGER RNA FOR BK VIRUS VP1 IN URINE 1 RUCHUANG DING, 2 MARA MEDEIROS, 2 DARSHANA DADHANIA, 2 THANGAMANI MUTHUKUMAR, 2 DAVID KRACKER, 2 JIN M. KONG, 2 SUSANNA R. EPSTEIN, 2 VIJAY K. SHARMA, 2 SURYA V. SESHAN, 3 BAOGUI LI, 2 AND MANIKKAM SUTHANTHIRAN 2,4 Background. Polyoma virus type BK (BKV) nephritis has emerged as an important cause of renal allograft dysfunction and graft failure. Its diagnosis is contin- gent on the invasive procedure of allograft biopsy. A noninvasive diagnostic test for BKV nephritis could improve clinical outcome. Methods. We obtained 25 urine specimens from 8 renal allograft recipients with biopsy-confirmed BKV nephritis, 31 samples from 28 recipients in whom BKV nephritis was excluded by allograft biopsy, and 74 specimens from 34 patients with stable allograft func- tion. RNA was isolated from the urinary cells and re- verse transcribed to complementary DNA. We de- signed gene-specific oligonucleotide primers and probes for the measurement of messenger RNA (mRNA) encoding BKV VP1 protein and a constitu- tively expressed 18S ribosomal RNA (rRNA) by real- time quantitative polymerase chain reaction. We ex- plored the hypothesis that BKV VP1 mRNA levels predict BKV nephritis. Results. The levels of BKV VP1 mRNA but not the levels of 18S rRNA predicted BKV nephritis. Analysis involving the receiver operating characteristic curve demonstrated that BKV nephritis can be predicted with a sensitivity of 93.8% and a specificity of 93.9% with the use of a cutoff value of 6.510 5 BKV VP1 mRNA copy number per nanogram of total RNA (P<0.00001). In the receiver operating characteristic curve analysis, the cal- culated area under the curve was 0.949 (95% confidence interval, 0.912 to 0.987, P<0.00001) for BKV VP1 mRNA levels and 0.562 (95% confidence interval, 0.417 to 0.708, P>0.2) for 18S rRNA. Conclusions. Measurement of BKV VP1 mRNA in uri- nary cells offers a noninvasive and accurate means of diagnosing BKV nephritis. Infections, especially of viral origin, remain a major cause of morbidity and mortality after organ transplantation (1,2). Recently, BK virus (BKV), a polyoma virus with a special predilection for the urinary tract, has emerged as a signifi- cant posttransplantation complication (3–6). Human poly- omavirus are members of the Polyomaviridae family and are approximately 40-nm icosahedrons with double-stranded, su- percoiled DNA of about 5130 base pairs (7,8). BKV and JC virus (JCV) were both named after the patients in whom they were identified: BKV from the urine of a renal transplant recipient with ureteral stenosis (9) and JCV from the brain tissue of a patient with progressive multifocal leukoenceph- alopathy (10). The nucleotide sequences of BKV and JCV show 75% homology. Primary BKV infection occurs during childhood, through the respiratory or gastrointestinal route. The primary infec- tion is usually asymptomatic but occasionally is associated with upper respiratory or urinary tract disease. After the primary infection, the virus remains latent in blood cells and in the urinary tract and can be reactivated in the immuno- compromised host. BKV infection results in hemorrhagic or nonhemorrhagic cystitis in bone marrow transplant recipi- ents, whereas renal allograft recipients present with hema- turia, ureteral stricture, and/or interstitial nephritis with graft dysfunction that can progress to graft loss (3–6,11–13). The diagnosis of active human polyomavirus infection is based on the examination of urine, direct visualization using electron microscopy, or by cytopathologic methods; by virus isolation; and by techniques that detect viral antigens or nucleic acids (3–6,14). Detailed evaluation of renal allograft core biopsy tissue is the most accurate way of diagnosing BKV nephritis, and the histologic features include character- istic epithelial intranuclear inclusion bodies, cytopathic changes in tubular cells, and interstitial cellular infiltrate. Evaluation of the allograft tissue by electron microscopy (15) or by immunohistochemical staining or both using antibodies against SV40 (a simian polyoma virus that shares 70% ho- mology with BKV and JCV) firmly establishes the diagnosis. A noninvasive and accurate procedure to diagnose active BKV infection in renal allograft recipients would be of consid- erable value. Indeed, polymerase chain reaction (PCR)-based assays have been developed to identify BKV DNA in plasma, serum, and peripheral blood leukocytes and in urine samples (16 –19). However, the ubiquitous nature of BKV is a frequent source of false-positive results in DNA-based tests (20 –23). The viral genome of BKV can be functionally divided into three regions: a noncoding regulatory region, an early region 1 This work was supported by an award (RO1 AI51652) from the National Institutes of Health and an award from Satellite Research/ Satellite Healthcare. Dr. Medeiros is supported by the Patronato del Hospital Infantil de M ´ exico “Federico G ´ omez” and CONACYT, Mexico. Dr. Dadhania is a recipient of the American Society of Transplantation Fujisawa Clinical Fellowship Award. Dr. Muthukumar is a recipient of a fellowship award from the International Society of Nephrology. Dr. Kracker is a recipient of a fellowship award from the National Kidney Foundation of New York/New Jersey. Dr. Kong’s present affiliation: Division of Nephrology, Department of Internal Medicine, Maryknoll Hospital, Pusan, Korea. 2 Division of Nephrology, Departments of Medicine and Trans- plantation Medicine, Weill Medical College of Cornell University, New York-Presbyterian Hospital, New York, NY. 3 Department of Pathology, Weill Medical College of Cornell Uni- versity, New York-Presbyterian Hospital, New York, NY. 4 Address correspondence to: M. Suthanthiran, M.D., 525 East 68th Street, Box 3, New York, NY 10021. E-mail: msuthan@ med.cornell.edu. Received 1 April 2002. Accepted 28 May 2002. 987 DOI: 10.1097/01.TP.0000032151.07990.1E